Bacterial infection of lung airways underlies some of the main complications of COPD, including acute exacerbations, which significantly impact disease progression and outcome. Furthermore, airway (and parenchymal) colonization by bacteria and viruses may synergize, amplify, or trigger pathways of tissue damage started by cigarette smoke, leading to airway inflammation with remodeling and alveolar destruction. The present application is centered on the concept that bacteria populations present in the lung of smokers interact with environmental stresses triggered by cigarette smoke and pollutants to cause lung injury, including lung inflammation and apoptotic cell death. To this end, we have relied on amplification and sequencing of the bacterial small subunit ribosomal RNA (SSU-rRNA) gene in a large number of phenotypically and clinically well-characterized COPD lung samples, allowing us to identify bacteria populations present in smokers' and COPD diseased lungs, notably of Novosphingobium ssp, in extensive preliminary studies leading to this application. Furthermore, we also detected Novosphingobium spp DNA in extracts of the Marlboro commercial cigarettes and in mouse lungs exposed to cigarette smoke. In this proposal, we hypothesize that the COPD lung harbors specific microbial communities, which segregate in specific lung compartments (airway vs. alveolus). In particular, we seek to demonstrate that Novosphingobium spp, identified in our ongoing microbiome studies, may be acquired through the inhalation of cigarette smoke and in the lung, trigger the activation of cytotoxic immunity against pulmonary cells associated with increased cytokine production in the setting of cigarette smoke exposure. Specific Aim 1 will demonstrate that human lungs exposed to cigarette smoke contain Novosphingobium spp, which are present in cigarettes and transmitted into hosts through cigarette smoke exposure. Using a sensitive qPCR, we will test whether levels of Novosphingobium spp DNA increase with COPD disease severity. Specific Aim 2 will determine the segregation of Novosphingobium spp and potentially relevant microbiota in anatomic lung compartments, such as the airways and alveolated tissue, in patients with COPD, smokers with minimal or mild disease, and in normal lungs using laser capture microdissection. Specific Aim 3 will address whether Novosphingobium infection of alveolar epithelial cells can lead to activation of NK, and/or CD8 T cells via enhanced expression of NKG2D ligands, leading to killing of infected alveolar epithelial cells and/or interferon-3 production as evidence of cell activation. This potentially high-impact proposal fulfills the premises of the R21 funding mechanism in that the elucidation of the presence and types of lung bacterial populations in different stages of COPD may reveal important insights into the pathobiology of the disease. Notably, our studies may indicate that the potential activation of innate and acquired immunity triggered by bacterial products and chronically injured lung cells and structures may trigger auto-immunity, which contribute to alveolar cell death and lung destruction.